scholarly journals Pattern of chick gene activation in chick erythrocyte heterokaryons.

1982 ◽  
Vol 95 (3) ◽  
pp. 885-892 ◽  
Author(s):  
S Linder ◽  
S H Zuckerman ◽  
N R Ringertz
Keyword(s):  
Hybrid Cell ◽  
Globin Gene ◽  
Gene Activation ◽  
Parental Cell ◽  
Mrna Levels ◽  
Cell Type ◽  
Globin Mrna ◽  
Two Dimensional Gel Electrophoresis ◽  
L6 Myoblast ◽  
Globin Synthesis

The reactivation of chicken erythrocyte nuclei in chick-mammalian heterokaryons resulted in the activation of chick globin gene expression. However, the level of chick globin synthesis was dependent on the mammalian parental cell type. The level of globin synthesis was high in chick erythrocyte-rat L6 myoblast heterokaryons but was 10-fold lower in chick erythrocyte-mouse A9 cell heterokaryons. Heterokaryons between chick erythrocytes and a hybrid cell line between L6 and A9 expressed chick globin at a level similar to that of A9 heterokaryons. Erythrocyte nuclei reactivated in murine NA neuroblastoma, 3T3, BHK and NRK cells, or in chicken fibroblasts expressed less than 5% chick globin compared with the chick erythrocyte-L6 myoblast heterokaryons. The amount of globin expressed in heterokaryons correlated with globin mRNA levels. Hemin increased beta globin synthesis two- to threefold in chick erythrocyte-NA neuroblastoma heterokaryons; however, total globin synthesis was still less than 10% that of L6 heterokaryons. Distinct from the variability in globin expression, chick erythrocyte heterokaryons synthesized chick constitutive polypeptides in similar amounts independent of the mammalian parental cell type. Approximately 40 constitutive chick polypeptides were detected in heterokaryons after immunopurification and two-dimensional gel electrophoresis. The pattern of synthesis of these polypeptides was similar in heterokaryons formed by fusing chicken erythrocytes with rat L6 myoblasts, hamster BHK cells, or mouse neuroblastoma cells. Three polypeptides synthesized by non-erythroid chicken cells but less so by embryonic erythrocytes were conspicuous in heterokaryons. Two abundant erythrocyte polypeptides were insignificant in non-erythroid chicken cells and in heterokaryons.

Globin synthesis in hybrid cells constructed by transplantation of dormant avian erythrocyte nuclei into enucleated fibroblasts

1981 ◽  
Vol 1 (12) ◽  
pp. 1163-1176
Author(s):  
J Bruno ◽  
N Reich ◽  
J J Lucas
Keyword(s):  
Ribonucleic Acid ◽  
Hybrid Cell ◽  
Globin Gene ◽  
Cell System ◽  
Nuclear Transplantation ◽  
Hybrid Cells ◽  
Two Dimensional Gel Electrophoresis ◽  
Messenger Ribonucleic Acid ◽  
Globin Chains ◽  
Globin Synthesis

The polypeptides synthesized by mature embryonic erythrocytes prepared from the peripheral blood of 14- to 15-day-old chicken embryos were analyzed by two-dimensional gel electrophoresis. Fewer than 200 species of polypeptides were detected; the major polypeptides made at this time were identified as the alpha A-, alpha D-, and beta-globin chains. The dormant erythrocyte nuclei were next reactivated to transcriptional competence by transplantation into enucleated mouse or chicken embryo fibroblasts, with frequencies of cytoplast renucleation of about 50 and 90%, respectively. Since large numbers of hybrid cells could be constructed, a biochemical analysis was possible. Electrophoretic analysis of the [35S]methionine-labeled polypeptides made in the hybrid cell types showed that polypeptides having the mobilities of only two (alpha A and alpha D) of the three major adult globin chains were made as major constituents of the hybrid cells. However, analysis of 14C-amino acid-labeled polypeptides revealed that a beta-like polypeptide that lacked methionine was also synthesized in large amounts. This polypeptide was tentatively identified as the early embryonic globin species rho. Globin synthesis was detected as early as 3 h after nuclear transplantation and as late as 18 h, the last time measured in these experiments. It appeared that globin polypeptides made at very early times were translated at least partially from chicken messenger ribonucleic acid introduced into the hybrid cells during fusion, whereas those made at later times were translated primarily from newly synthesized globin messenger ribonucleic acid. The potential usefulness of this hybrid cell system in analyzing mechanisms regulating globin gene expression is discussed.

scholarly journals Globin synthesis in hybrid cells constructed by transplantation of dormant avian erythrocyte nuclei into enucleated fibroblasts.

1981 ◽  
Vol 1 (12) ◽  
pp. 1163-1176 ◽  
Author(s):  
J Bruno ◽  
N Reich ◽  
J J Lucas
Keyword(s):  
Ribonucleic Acid ◽  
Hybrid Cell ◽  
Globin Gene ◽  
Cell System ◽  
Nuclear Transplantation ◽  
Hybrid Cells ◽  
Two Dimensional Gel Electrophoresis ◽  
Messenger Ribonucleic Acid ◽  
Globin Chains ◽  
Globin Synthesis

The polypeptides synthesized by mature embryonic erythrocytes prepared from the peripheral blood of 14- to 15-day-old chicken embryos were analyzed by two-dimensional gel electrophoresis. Fewer than 200 species of polypeptides were detected; the major polypeptides made at this time were identified as the alpha A-, alpha D-, and beta-globin chains. The dormant erythrocyte nuclei were next reactivated to transcriptional competence by transplantation into enucleated mouse or chicken embryo fibroblasts, with frequencies of cytoplast renucleation of about 50 and 90%, respectively. Since large numbers of hybrid cells could be constructed, a biochemical analysis was possible. Electrophoretic analysis of the [35S]methionine-labeled polypeptides made in the hybrid cell types showed that polypeptides having the mobilities of only two (alpha A and alpha D) of the three major adult globin chains were made as major constituents of the hybrid cells. However, analysis of 14C-amino acid-labeled polypeptides revealed that a beta-like polypeptide that lacked methionine was also synthesized in large amounts. This polypeptide was tentatively identified as the early embryonic globin species rho. Globin synthesis was detected as early as 3 h after nuclear transplantation and as late as 18 h, the last time measured in these experiments. It appeared that globin polypeptides made at very early times were translated at least partially from chicken messenger ribonucleic acid introduced into the hybrid cells during fusion, whereas those made at later times were translated primarily from newly synthesized globin messenger ribonucleic acid. The potential usefulness of this hybrid cell system in analyzing mechanisms regulating globin gene expression is discussed.

Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression

1985 ◽  
Vol 5 (4) ◽  
pp. 649-658
Author(s):  
G M Veldman ◽  
S Lupton ◽  
R Kamen
Keyword(s):  
Dna Replication ◽  
Globin Gene ◽  
Mrna Levels ◽  
Beta Globin ◽  
Transcriptional Enhancer ◽  
Globin Mrna ◽  
Enhancer Region ◽  
Sequence Elements ◽  
Multiple Copies ◽  
A Site

Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the beta-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the beta-globin gene. We observed a correlation between the number of inserted 26-mers and the level of beta-globin RNA expression.

scholarly journals Diagnosis of thalassemia using cDNA amplification of circulating erythroid cell mRNA with the polymerase chain reaction [published erratum appears in Blood 1992 Jun 15;79(12):3397]

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2433-2437 ◽  
Author(s):  
SZ Huang ◽  
GP Rodgers ◽  
FY Zeng ◽  
YT Zeng ◽  
AN Schechter
Keyword(s):  
Polymerase Chain Reaction ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Beta Globin ◽  
Chain Reaction ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Polymerase Chain ◽  
Alpha Beta

Abstract We have developed a technique to diagnose the alpha- and beta- thalassemia (thal) syndromes using the polymerase chain reaction to amplify cDNA copies of circulating erythroid cell messenger RNA (mRNA) so as to quantitate the relative amounts of alpha-, beta-, and gamma- globin mRNA contained therein. Quantitation, performed by scintillation counting of 32P-dCTP incorporated into specific globin cDNA bands, showed ratios of alpha/beta-globin mRNA greater than 10-fold and greater than fivefold increased in patients with beta 0- and beta (+)- thal, respectively, as well as a relative increase in gamma-globin mRNA levels. Conversely, patients with alpha-thalassemia showed a decreased ratio of alpha/beta-globin mRNA proportional to the number of alpha- globin genes deleted. This methodology of ascertaining ratios of globin mRNA species provides a new, simplified approach toward the diagnosis of thalassemia syndromes, and may be of value in other studies of globin gene expression at the transcription level.

5-Azacytidine Induction of Human γ-Globin Gene Expression: Experimental Evaluation of Current Models.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1581-1581
Author(s):  
Rodwell Mabaera ◽  
Christine Richardson ◽  
Sarah Conine ◽  
Christopher H. Lowrey
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Maximal Response ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Potent Inducer ◽  
Cellular Dna ◽  
Globin Gene Expression

Abstract 5-Azacytidine (5-Aza) was demonstrated to be a potent inducer of human fetal globin gene expression more than 20 years ago. More recently, 5-Aza-2-deoxycytidine has been shown to have similar properties. Since the 1980’s there have been two predominant hypotheses to explain the action of these agents. The first is based on the observation that these, and several other active inducing agents, are cytotoxic to differentiating erythroid cells and that drug treatment alters the kinetics of erythroid differentiation. This has been proposed to result in prolonged expression of the γ-globin genes which are normally expressed only early in differentiation. The second is based on the observation that both agents are DNA methyltransferase inhibitors and are presumed to cause demethylation of cellular DNA including the γ-globin gene promoters leading to activation of the genes. These two models lead to specific predictions that we have evaluated using an in vitro erythroid differentiation system. In this system, human adult CD34+ cells are cultured in SCF, Flt3 ligand and IL-3 for 7 days and then switched to Epo for 14 days. This results in an exponential expansion of erythroid cells. As has been described for normal human differentiation, these cells express small amounts of γ-globin mRNA early in differentiation followed by a much larger amount of β-globin mRNA. HPLC at the end of the culture period shows 99% HbA and 1% HbF. Treatment of cultures on a daily basis with 5-Aza starting on day 10 results in dose dependent increases in γ-globin mRNA, Gγ- and Aγ-chain production and HbF. The cytotoxicity model predicts that γ-globin expression will be prolonged to later in differentiation - and this is seen. However, a daily 5-Aza dose of 300 nM, which produces ~80% of the maximal response in γ-globin mRNA and HbF, has no effect on cell growth or differentiation kinetics. This argues against the toxicity model. We next examined the effect of 5-Aza on γ-globin promoter methylation using the bisulfite method. We studied CpGs at −344, −252, −162, −53, −50, +6, +19 and +50 relative to the start site. For untreated controls, all of the sites are nearly 100% methylated at day 1. By day 3, the upstream sites become ~50% methylated except the −53 CpG which was <20%. This pattern persisted at day 10. By day 14 the promoters had become largely remethylated. For cells treated with 5-Aza starting on day 10, there was no change in the levels of methylation seen on days 1,3 and 10, but at day 14 the low levels of upstream methylation persisted - just as γ-globin expression does. However, in both treated and untreated cells, down-stream CpG sites were highly methylated at all time points. This suggests that γ promoter demethylation may be due to a local and not a generalized effect of 5-Aza on cellular DNA methylation. We also made two unexpected observations. At a 300nM dose of 5-Aza, γ-globin mRNA is ~doubled while β-globin mRNA levels are ~halved - indicating that 5-Aza not only induces γ-globin expression also suppresses β-globin. Also despite only a doubling in γ-globin mRNA, there was an ~50-fold increase in HbF, from ~1% to more than 50%, while total per cell Hb levels were unchanged. Neither of these results are easily explained by current models of γ-globin gene induction. Our results raise the possibility that mechanisms beyond cytotoxicity and generalized DNA demethylation may be responsible for pharmacologic induction of γ-globin mRNA and HbF.

Gene Expression Profiles Involved in γ to β Globin Gene Switching during Erythroid Maturation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 820-820
Author(s):  
Wei Li ◽  
Betty S. Pace
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Globin Gene ◽  
Gene Expression Profiles ◽  
Phase System ◽  
Mrna Levels ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Gene Switching ◽  
Globin Gene Expression

Abstract The design and evaluation of therapies for sickle cell disease (SCD) rely on our understanding of hemoglobin accumulation during erythropoiesis and sequential globin gene expression (ε → Gγ → Aγ → δ → β) during development. To gain insights into globin gene switching, we completed time course micorarray analyses of erythroid progenitors to identify trans-factors involved in γ gene activation. Studies were completed to map the pattern of γ and β globin gene expression in progenitors grown from normal peripheral blood mononuclear cells. We compared cells grown in a 2-phase (phase 1, d0-6: SCF, IL-3, IL-6, and GM-CSF and phase 2, d7-25: SCF and EPO) vs. 1-phase (d0-34: SCF, IL-3, and EPO) liquid culture system. From day 0 to 34 in either system cell viability remained &gt;99%. Total RNA was isolated using Trizol and column cleanup (Qiagen). Globin mRNA levels were measured at 2–3 day intervals by quantitative PCR (qPCR). In the 2-phase system γ-globin mRNA&gt;β-globin mRNA up to d14, 4 days of approximately equal expression then β mRNA &gt; γ mRNA by d20. By contrast, in 1-phase studies there was a rapid switch around d20(see graph). We speculate that this difference may be due to the early addition of EPO on d0 therefore we continued our detailed analysis in this system. To confirm that our in vitro system recapitulates in vivo gene expression patterns, we completed studies to ascertain Gγ - vs. Aγ globin mRNA levels. The normalized Gγ:Aγ ratio decreased from ~3:1 on d7 to ~1:1 by d34; These findings were confirmed using two sets of Gγ and Aγ globin primers. We concluded that the 1-phase system recapitulated normal γ/β globin switching and that gene profiling studies to identify the trans-factor involved in switching mechanisms were feasible. We used Discover oligo chips (ArrayIt, Sunnyvale, CA) containing 380 human genes selected from 30 major functional groups including hematopoiesis. To aide interpretation of chip data, cell populations were rated morphologically using Giemsa stained cytospin preps. From d16 on we observed an increase in late erythroid progenitors (normoblasts) from 1% to 71% by d31. After verifying RNA quality by gel inspection of ribosomal molecules, we prepared Cy3 and Cy5 probes for early and late time-point RNA samples respectively. Chip analysis was performed at several time points but d0/21, d7/21, and d21/28 were most informative. Based on Axon GenePixPro 6.0 and Acuity 4.0 software analysis we found the following genes with &gt;1.5-fold change in expression profile (shown as down-regulated/up-regulated genes): d0/21: 33/73, d7/21: 13/25, and d21/28:35/26. Principal component analysis (PCA), hierarchical clusters and self organizing maps were constructed. Gene profiles were correlated with the γ/β switching curve using d7 (γ &gt;β), d21 (γ ~ β), and d28 (γ &lt;β) data. Hematopoietic dataset analysis at d21 revealed 4 candidate γ-globin gene activators including v-myb, upsteam binding transfactor -RNApol1 and 2 zinc finger proteins. Analysis of a d28 dataset revealed 12 proteins involved in γ-globin gene silencing including IL-3, SCF, MAPKKK3, v-raf-1, ATF-2, and glucocorticoid receptor DNA binding factor 1 among others. Gene expression profiles will be validated using qPCR and promising candidates will be tested by forced expression in transient and stable reporter systems. Figure Figure

Differences in Fetal Hemoglobin Induction in Sickle Cell Disease and beta-Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 555-555 ◽  
Author(s):  
Hassana Fathallah ◽  
Ali Taher ◽  
Ali Bazarbachi ◽  
George F. Atweh
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Mrna Levels ◽  
Globin Genes ◽  
Thalassemia Intermedia ◽  
Cell Disease ◽  
Globin Mrna ◽  
Globin Gene Expression

Abstract A number of therapeutic agents including hydroxyurea, butyrate and decitabine have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia. As a first step towards understanding the molecular basis of the different clinical responses to these agents, we have studied the mechanisms of induction of fetal hemoglobin (HbF) by butyrate in BFU-E derived cells from 5 patients with SCD and 9 patients with β-thalassemia intermedia. Exposure to butyrate resulted in a dose-dependent augmentation of γ-globin mRNA levels in erythroid cells from patients with SCD. In contrast, induction of γ-globin expression in erythroid cells from patients with β-thalassemia intermedia was only seen at a high concentration of butyrate. The increase in γ-globin mRNA levels in patients with SCD and β-thalassemia intermedia was associated with opening of the DNA structure as manifested by decreased DNA methylation at the γ-globin promoters. Interestingly, butyrate exposure had markedly different effects on the expression of the β- and α-globin genes in the two categories of patients. Butyrate decreased the level of β-globin mRNA in 4 out of 5 patients with SCD (P = 0.04), while in β-thalassemia the levels of β-globin mRNA did not change in 7 patients and decreased in 2 patients after butyrate exposure (P = 0.12). Thus in patients with SCD, the effects of the induction of the γ-globin gene on the γ/(β+γ) mRNA ratios were further enhanced by the butyrate-mediated decreased expression of the β-globin gene. As a result, γ/(β+γ) mRNA ratios increased in all patients with SCD, with a mean increase of 31% (P = 0.002). In contrast, butyrate increased γ/(β+γ) mRNA ratios only in 4 out of 9 patients with β-thalassemia, with a more modest mean increase of 12% (P = 0.004). Interestingly, the decreased β-globin expression in patients with SCD was associated with closing of the DNA configuration as manifested by hypermethylation of DNA at the promoter of the β-globin gene while methylation of the same promoter did not change following butyrate exposure in patients with β-thalassemia intermedia. More surprisingly, the expression of the α-globin genes increased following butyrate exposure in 4 out of 9 patients with β-thalassemia, while the levels of α-globin mRNA decreased in 4 out of 5 patients with SCD. As a result, the favorable effects of the butyrate-induced increase in γ-globin gene expression on the α: non-α mRNA imbalance in patients with β-thalassemia intermedia were partly neutralized by the corresponding increase in α-globin gene expression. These differences may explain, at least in part, the more favorable effects of inducers of HbF in SCD than in β-thalassemia. Further studies are necessary to fully understand the molecular bases of the different responses to agents that induce HbF in patients with these disorders.

Globin Genes Induction by Nitric Oxide of Stromal Cell Origin.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4102-4102
Author(s):  
Vladan P. Cokic ◽  
Bojana B. Beleslin-Cokic ◽  
Constance Tom Noguchi ◽  
Alan N. Schechter
Keyword(s):  
Progenitor Cells ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Globin Genes ◽  
Globin Mrna ◽  
Erythroid Progenitor ◽  
Gamma Globin ◽  
Erythroid Progenitor Cells ◽  
Globin Gene Expression

Abstract We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). Here we report that co-culture of human bone marrow endothelial cells with erythroid progenitor cells induced gamma-globin mRNA expression (1.8 fold), and was further elevated (2.4 fold) in the presence of hydroxyurea (40 μM). Based on these results, NOS-dependent stimulation of NO levels by bradykinin and lipopolysaccharide has been observed in endothelial (up to 0.3 μM of NO) and macrophage cells (up to 6 μM of NO), respectively. Bradykinin slightly increased gamma-globin mRNA levels in erythroid progenitor cells, but failed to increase gamma-globin mRNA levels in endothelial/erythroid cell co-cultures indicating that stimulation of endothelial cell production of NO alone is not sufficient to induce gamma-globin expression. In contrast, lipopolysaccharide and interferon-gamma mutually increased gamma-globin gene expression (2 fold) in macrophage/erythroid cell co-cultures. In addition, hydroxyurea (5–100 μM) induced NOS-dependent production of NO in human (up to 0.7 μM) and mouse macrophages (up to 1.2 μM). Co-culture studies of macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to 3 fold) in the presence of hydroxyurea (20–100 μM). These results demonstrate a mechanism by which hydroxyurea may induce globin genes and affect changes in the phenotype of hematopoietic cells via the common paracrine effect of bone marrow stromal cells.

scholarly journals The Effect of the Soluble Guanylyl Cyclase Stimulator Olinciguat on ƴ-Globin Gene Induction in K562 Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1078-1078 ◽  
Author(s):  
Joy Miyashiro ◽  
Asha Pant ◽  
Boris Tchernychev ◽  
Todd G Milne ◽  
Mark G Currie ◽  
...  
Keyword(s):  
Cell Line ◽  
Guanylyl Cyclase ◽  
Mode Of Action ◽  
Globin Gene ◽  
Soluble Guanylyl Cyclase ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Mrna Levels ◽  
Globin Mrna ◽  
No Signaling

Abstract Induction of fetal hemoglobin (HbF: α2ƴ2) is a recognized mode of action of hydroxyurea, the sickle cell disease (SCD) standard of care in SCD, and has been shown to prevent red blood cell (RBC) sickling. Discovery of novel HbF inducers is underway and several therapeutics with the potential to increase HbF expression are currently at different stages of preclinical and clinical development. Soluble guanylyl cyclase (sGC) is a heterodimeric heme-containing enzyme whose catalytic activity is regulated by nitric oxide (NO). Binding of NO to heme activates the catalytic domain of sGC, enabling synthesis of the second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate. sGC stimulators are small molecules that synergize with NO to boost signaling via the NO-sGC-cGMP pathway. This signaling pathway is involved in the regulation of many physiologic processes including inflammation, fibrosis, and blood flow. Perhaps less well-known, cGMP-mediated signaling has also been implicated in the regulation of the gene encoding the ƴ-globin subunit of fetal hemoglobin (Modulation of NO signaling by sGC stimulation, therefore, has the therapeutic potential to target the complex pathology of SCD at multiple levels. In this study, we focused on one potential mode of action of sGC stimulation-increasing HbF expression. We characterized the effects of the sGC stimulator olinciguat on ƴ-globin gene expression. Olinciguat is currently being investigated for the treatment of patients with SCD in a Phase II STRONG-SCD study (NCT03285178). The effect of olinciguat treatment on ƴ-globin mRNA levels was studied in the K562 erythroleukemic cell line. For short-term (8 hours) treatment with olinciguat, K562 cells were maintained in a serum-free media. For long-term (4 and 7 days) treatment, cell culture media contained 1% fetal bovine serum. Hydroxyurea was used as a positive control. Levels of ƴ-globin mRNA were expressed relative to mRNA levels of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. K562 cells were treated for 8 hours with increasing concentrations of olinciguat (0.01, 0.1, 1, and 10 µM). Treatment of K562 cells with 0.1, 1, and 10 µM of olinciguat increased ƴ-globin mRNA levels by 1.43±0.08-, 1.37±0.06-, and 1.47±0.06-fold (mean±SEM), respectively. For comparison, 8 hours of treatment with hydroxyurea (800 µM) increased ƴ-globin mRNA levels by 1.25±0.03-fold. When K562 cells were cultured in the presence of olinciguat for 4 days, significant (P<0.05) induction of ƴ-globin mRNA levels was observed at 1 and 10 µM (1.13±0.03- and 1.55±0.09-fold, respectively). Induction of ƴ-globin mRNA following 4 days of incubation with hydroxyurea (800 µM) was 2.38±0.2-fold. The effects of hydroxyurea and olinciguat on ƴ-globin mRNA levels were compared following 7 days of incubation with the compounds. After 7 days of treatment of K562 cells with 0.1, 1, 3, and 10 µM of olinciguat, ƴ-globin mRNA levels were increased by 1.83±0.19-, 1.66±0.09-, 2.4±0.06-, and 2.9±0.33-fold, respectively. Treatment with 50- and 800-µM hydroxyurea increased levels of ƴ-globin mRNA by 2.33±0.15- and 3.8±0.56-fold, respectively. In conclusion, the sGC stimulator olinciguat increased the expression of mRNA for the ƴ-globin subunit of fetal hemoglobin in the erythroleukemic K562 cell line. This finding indicates that amplifying NO signaling by stimulating sGC may increase HbF expression, thereby preventing pathologic RBC sickling; this extends the potential therapeutic utility of olinciguat in SCD. Finally, the ability of olinciguat to induce HbF in SCD patients will be assessed in the ongoing Phase II STRONG-SCD study (NCT03285178). Disclosures Miyashiro: Ironwood Pharmaceuticals: Employment. Pant:Ironwood Pharmaceuticals: Employment. Tchernychev:Ironwood Pharmaceuticals: Employment, Equity Ownership. Milne:Ironwood Pharmaceutics, Inc: Employment. Currie:Ironwood Pharmaceuticals: Employment. Graul:Ironwood Pharmaceuticals, Inc: Employment. Masferrer:Ironwood Pharmaceuticals, Inc: Employment.

Quantitative PCR analysis of HbF inducers in primary human adult erythroid cells

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 863-869 ◽  
Author(s):  
Reginald D. Smith ◽  
Jin Li ◽  
Constance T. Noguchi ◽  
Alan N. Schechter
Keyword(s):  
Quantitative Pcr ◽  
Butyric Acid ◽  
Trichostatin A ◽  
Quantitative Polymerase Chain Reaction ◽  
Globin Gene ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Human Adult

The development and evaluation of drugs to elevate fetal hemoglobin in the treatment of the genetic diseases of hemoglobin would be facilitated by the availability of reliable cell assays. We have used real-time, quantitative polymerase chain reaction (PCR) analyses of globin messenger RNA (mRNA) levels in a biphasic, erythropoietin-dependent primary culture system for human adult erythroid cells in order to assay compounds for their ability to modulate levels of adult (β) and fetal (γ) globin mRNA. Complementary DNA synthesized from total RNA extracted at timed intervals from aliquots of cells were assayed throughout the period that the culture was studied. γ-globin mRNA levels were found to be much lower (less than 1%) than β-globin mRNA levels. At concentrations of agents chosen for minimal effect on cell division, we find that the 3 drugs studied, 5-azacytidine (5μmol/L), hydroxyurea (40μmol/L), and butyric acid (0.5mmol/L), significantly increase γ-globin mRNA levels. Interestingly, hydroxyurea also had a small stimulatory effect on β-globin mRNA levels, while butyric acid caused a twofold inhibition of β-globin mRNA levels, and 5-azacytidine had little effect on β-globin mRNA levels. The net result of all 3 drugs was to increase the γ/(γ + β) mRNA ratios by threefold to fivefold. These data suggest that the mechanism is distinct for each drug. The profile of butyric-acid–induced changes on globin gene expression is also quite distinct from changes produced by trichostatin A, a known histone deacetylase inhibitor. Quantitative PCR analyses of human erythroid cells should prove useful for studying the mechanism(s) of action of known inducers of γ-globin and identifying new drug candidates.

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